Stage- and tissue-specific expression of the genes encoding calliphorin,the major larval serum protein of Calliphora vicina

Summary The stage- and tissue-specific biosynthesis of calliphorin was analysed during the development of the blowfly, Calliphora vicina. Western blot analyses show that the protein is not present in eggs, whereas it can be detected in fat body, brain, imaginai disk, salivary gland and epidermis throughout all postembryonic stages, including the adult one. By Northern analysis a unique 2.6 kb mol.wt. mRNA coding for calliphorin is identified exclusively in the fat body tissue of larvae, pupae and adults. Hybridization experiments of in vivo labelled poly(A)⁺ RNA with filter-bound calliphorin genes indicate that the genes are transcribed until pupariation. However, the translation of the calliphorin mRNA stops at the end of the feeding stage, as shown by [³⁵S]-methionine incorporation.     

The isolation and properties of the protein calliphorin

1. A procedure for the isolation of the protein calliphorin from larvae and pupae of the blowfly Calliphora erythrocephala is described. 2. The calliphorin preparation shows a single component in the ultracentrifuge at pH6.3 and gives a single band when stained for protein after agar-gel or starch-gel electrophoresis at pH6.3 or 8.6. Immunoelectrophoresis yields only one arc, associated with the stained spot, to a rabbit antiserum known to react with 13 other soluble components of Calliphora pupae. 3. Calliphorin has s⁰_20,w 19.4S, D⁰_20,w 3.25×10⁻⁷cm²·s⁻¹ and f/f₀ 1.22, indicating a molecular weight of 528000 and a compact symmetrical structure. The molecular weight determined by the meniscus-depletion sedimentation-equilibrium method is 529000. 4. In 6.2m-guanidine hydrochloride calliphorin dissociates into six components each with a molecular weight of about 87000. Calliphorin reversibly dissociates into components with sedimentation coefficients of about 7S as the pH is raised progressively above pH6.5. 5. Calliphorin has an unusually high tyrosine and phenylalanine content (442 and 400mol/mol of protein respectively), a relatively high methionine content (162mol/mol of protein) and very little cystine or cysteine (18mol/mol of protein). The E₂₈₀/E₂₅₀ ratio is 3.2. The pure protein contains 0.4–0.5% carbohydrate. 6. When examined in the electron microscope by the negative staining technique the protein is seen to consist of particles which are right prisms, being 105Å wide and 65Å high, rectangular in side view and curvilinear equilateral triangles in surface view.     

Studies on gastric mucoproteins. The production of radioactive mucoproteins by pig gastric mucosal scrapings in vitro

1. Optimum conditions, including the effect of media of different pH values, were determined for the incorporation of radioactive precursors into mucoproteins by pig gastric mucosa in vitro. 2. Mucosal scrapings incorporated radioactivity from [U-¹⁴C]-glucose and from [G-³H]threonine or [G-³H]serine solely into the carbohydrate and protein portions respectively of the mucoprotein molecules. 3. Of the radioactive mucoprotein 22% was water-soluble and up to 80% of the remainder was soluble in other solvents. 4. Pronase was the most successful proteolytic enzyme tested for making the mucoprotein water-soluble, up to 94% dissolving after digestion. 5. The Pronase digestion products of the mucoproteins were separated from protein by equilibrium-density-gradient centrifugation in a CsCl gradient. 6. These Pronase-digested mucoproteins were further fractionated on Sepharose 4B and the isolated fractions analysed by chemical and sedimentation-velocity methods. 7. Pronase digestion and solvent extraction of mucosal scrapings labelled with ¹⁴C in the carbohydrate and ³H in the protein showed that one type of mucoprotein was the only non-diffusible biosynthetic product of the scrapings in vitro, and that this mucoprotein was the only mucoprotein constituent of the water-soluble and water-insoluble mucus.     

Identification and molecular analysis of a multigene family encoding calliphorin, the major larval serum protein of Calliphora vicina

A library of Calliphora vicina genomic DNA was constructed in the λEMBL3 vector and screened for recombinant phages containing chromosomal segments encoding calliphorin, the major larval serum protein (LSP) of Calliphora. A large series of recombinants hybridizing with in vitro labelled poly(A)⁺ RNA from Calliphora larval fat bodies and with specific probes derived from the LSP-1 genes of Drosophila melanogaster was isolated. Five of these phages, chosen at random, were shown by hybrid selection to retain calliphorin mRNA specifically. Eleven calliphorin mRNA-homologous regions were located on restriction maps of these phages by hybridization with 5' end-labelled poly(A)⁺ RNA from Calliphora larval fat bodies. Each phage contains at least two calliphorin genes arranged in direct repeat orientation and seperated by 3.5–5 kb intergenic regions. The genes display similar but not identical restriction patterns. Filter hybridization and heteroduplex analysis indicate that they share a detectable homology with the LSP-1β gene of D. melanogaster. Whole genome Southern analysis showed that these genes belong to a large family of closely related calliphorin genes which were found by in situ hybridization to polytene chromosomes of trichogen cells to be clustered in region 4a of chromosome 2 of Calliphora vicina.     

Calliphorin,a major protein of the blowfly: Correlation between the amount of protein,its biosynthesis,and the titer of translatable calliphorin-mRNA during development

The amount of calliphorin, its biosynthesis, and the levels of translatable calliphorin-mRNA have been determined during the postembryonic development of Calliphora vicina R.-D. The amount of calliphorin increases in early third-instar larvae, reaching maximal levels in 6-day-old animals. It continuously decreases during late larval and pupal development to approximately one-half of the maximal levels and abruptly sinks during eclosion. The biosynthesis of calliphorin takes place only in 3- to 5-day-old larvae. Poly(A)⁺-RNA has been translated into proteins in a wheat germ cell-free system. Calliphorin-mRNA can be detected in 3- to 7-day-old larvae; maximal concentrations are observed in 4- and 5-day-old animals. No calliphorin-mRNA can be detected in prepupae, pupae, or imagos. The biosynthesis of calliphorin in blowfly larvae stops before a decrease of translatable calliphorin-mRNA is observed. This finding raises the question of the mechanism of in vivo inactivation of this specific mRNA.     

Rates of ribosomal protein and total protein synthesis during Drosophila early embryogenesis

Embryos at various stages of early development from 1.5 to 5 hr after oviposition were made permeable with octane and labeled for 1 hr with [³H]phenylalanine. Measurements of the rate of incorporation of [³H]phenylalanine into ribosomal proteins and total protein were made using these synchronized Drosophila embryos. The rate of synthesis of those ribosomal proteins incorporated into ribosomes increases until 3 to 4 hr after fertilization (550 pg/embryo-hr) then declines later in embryonic development. The rate of total protein synthesis is maximal as early during embryonic development as could be measured. During the period between 1.5 and 2.5 hr after fertilization this rate is 9.4 ng/embryo-hr and then also declines. The synthesis of ribosomal proteins accounts for a substantial portion (4.5%–8.9%) of total protein synthesis in early embryos. These results indicate that ribosome formation is a significant activity during the earliest stages of Drosophila development.     

ROLE OF RIBONUCLEASE ACTION IN PHENYLALANINE-INDUCED DISAGGREGATION OF RAT CEREBRAL POLYRIBOSOMES

—l -phenylalanine (1 mg/g body wt) or physiological saline (0.9% NaCl) was given intraperitoneally to infant (7-day old), immature (14-day old), and adult (42-day old) rats. The state of ribosomal aggregation was determined in the cerebral postmitochondrial supernatant and purified polyribosome fractions prepared in the presence of rat liver ribonuclease inhibitor. Polyribosomes isolated from cerebral cortices of infant and immature rats 30 or 60 min after administration of phenylalanine were partially disaggregated, whereas the state of aggregation of polyribosomes from mature cerebrum was unchanged. In contrast, little or no evidence of phenylalanine-induced polyribosome disruption was noted in the postmitochondrial supernatant fractions, from which the cerebral polyribosomes were prepared, in any of the animals. Omission of the ribonuclease inhibitor resulted in polyribosome disaggregation in the postmitochondrial supernatant fractions prepared from saline-treated as well as phenylalanine-treated infant rats, but the disruption was more profound in the latter group. Ribonuclease activities in cerebral postmitochondrial supernatant preparations from infant and immature rats were higher than the corresponding values in preparations from adult animals. In addition, the administration of phenylalanine resulted in increases in ribonuclease activities in cerebral postmitochondrial supernatant preparations from the younger animals, but had no effect on these activities in adult animals. These results suggest that alterations in structure and function of polyribosomes from the infant rat cerebrum following a loading dose of phenylalanine were related to exposure of the polyribosomes during isolation to elevated activities of cerebral ribonucleases resulting from this treatment. This hypothesis was supported by the finding that phenylalanine treatment had no effect on the incorporation in vivo of intracisternally-administered radioactive lysine into total, soluble or ribosomal protein of infant cerebrum. However, when cerebral ribosomal RNA was differentially labelled in phenylalanine-treated and saline-treated infant rats by the intracisternal administration of [³H] or [¹⁴C]uridine, and polyribosome fractions were then prepared from the pooled cerebral cortices of both groups, radioactive ribosomes derived from saline-treated rats were more highly aggregated than those derived from phenylalanine-treated animals. It is concluded that gross alterations in cerebral polyribosome structure and function do not occur in vivo in young rats given a large amount of phenylalanine intraperitoneally. However, this treatment, in addition to increasing ribonuclease activity in cerebral cell-free preparations, also sensitizes cerebral polyribosomes to subsequent breakdown upon exposure to ribonucleases during isolation.     

Metabolism of phenylalanine and tyrosine in tobacco cell lines resistant and sensitive to p-fluorophenylalanine

Chromatography of soluble polyphenols of p-fluorophenylalanine-sensitive and -resistant tobacco cells revealed that the 10-fold increased level found in the resistant line was largely due to the accumulation of two unidentified polyphenols. The uptake of Phe-[U-¹⁴C] and Tyr-[U-¹⁴C] by the resistant line was ca 10 % that by the sensitive line. About 90 % of the phenylalanine-[¹⁴C] which was taken up by both cell lines could be accounted for as free phenylalanine in protein, soluble polyphenols or CO₂. The fate of Tyr-[¹⁴C] was similar to that of phenylalanine except that the incorporation was into insoluble polymeric forms of polyphenols rather than into soluble polyphenols. The resistant line incorporated 9-times more phenylalanine-[¹⁴C] into soluble polyphenols than did the sensitive line. The different ¹⁴C-aromatic amino acid accumulation and incorporation patterns noted with the two cell lines indicates that there are different active pools. Differential uptake rates by the two cell lines might affect the distribution of the absorbed amino acid among the different pools.     

Effects of farnesyl methyl ether on the haemolymph and ovarian proteins of the tent caterpillar, Malacosoma americanum

The mode of antigonadotropic action of farnesyl methyl ether (FME) on Malacosoma americanum was investigated by studying the haemolymph and ovarial proteins during vitellogenesis. Low doses of FME which permitted apparently normal adult development but inhibited ovarian development were used.As indicated by the incorporation of ¹⁴C-glycine, FME treatment had no effect on the protein biosynthetic activity by the fat body. However, it resulted in significant accumulation of several haemolymph proteins apparently caused by their reduced uptake by the ovarioles.Radioassay of individual proteins revealed that all the haemolymph proteins had incorporated ¹⁴C-glycine. However, proteins G and H, and to some extent protein B of the treated females, showed significantly higher incorporation of the labelled glycine. It is concluded that the antigonadotropic action of the JH mimic was due to reduced incorporation of various sex-specific and non-specific haemolymph proteins into the ovarioles.     

Biosynthesis of hydroxyphaseollin and related isoflavanoids in disease-resistant soybean hypocotyls

Phenylalanine-U-¹⁴C and isoliquiritigenin-9-¹⁴C were readily incorporated into the antifungal pterocarpan hydroxyphaseollin in soybean hypocotyls that were inoculated with incompatible strains of the phytopathogenic fungus Phytophthora megasperma var. sojae. Hydroxyphaseollin accounted for over half of the phenylalanine and isoliquiritigenin incorporated into ethyl acetate soluble compounds. Daidzein, coumestrol, and sojagol were identified as major compounds which accumulated coordinately with hydroxyphaseollin and contained significant amounts of radioactivity from the labelled isoflavanoid precursors. Hydroxyphaseollin was not present in healthy soybean plants and was not detected until ca. 16 hr after inoculation with the fungus. The pterocarpan then accumulated rapidly between 16 and 48 hr after inoculation, while the greatest accumulations of daidzein, coumestrol, and sojagol occurred between 48 and 72 hr after inoculation, when hydroxyphaseollin accumulation had ceased. Although soybean hypocotyls contained the anthocyanin malvin, neither this compound nor any other flavone pathway product was observed to accumulate after fungus-inoculation. The results therefore indicate that the accumulation of hydroxyphaseollin in fungus-inoculated soybean hypocotyls involves the activation of isoflavanoid biosynthesis with 'direction' of metabolic intermediates to biosynthesis of the pterocarpan.     

Metabolism of [6-14C]orotate by shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus

Incorporation of radioactivity from [6-¹⁴C]orotate into the pyrimidine constituents of shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus was examined with special reference to the unusual pyrimidine constituents. With each species, although 80% of the orotate supplied was catabolized to β-alanine, all the pyrimidine derivatives became radioactively labelled. With Pisum, the major part of the radioactivity incorporated into pyrimidines was located in UMP and the uracil derivatives, including the uracilyl amino acids willardiine and isowillardiine. With Phaseolus, UMP and the uracil derivatives were again the major radioactive products; incorporation of radioactivity into 5-ribosyluracil (pseudouridine), which accumulates in Phaseolus tissues, was comparable to the incorporation into orotidine and twice that found in cytidine. Lathyrus incorporated a substantially larger part of the presented [6-¹⁴C] orotate into pyrimidine derivatives than did the other two species. CMP was the most highly radioactive product, followed next by lathyrine and UMP. Surprisingly, 20% of the total radioactivity incorporated into pyrimidines by Lathyrus was located in the pyrimidine amino acid lathyrine. This confirms previous evidence that lathyrine is essentially a product of the orotate pathway. The overall recovery of radioactivity in all three species was 93–95%. The data emphasize the necessity of including the less common pyrimidine constituents, as well as the common ones, in quantitative studies of pyrimidine metabolism in plants.     

Proline biosynthesis in a proline-accumulating barley mutant

A barley (Hordeum vulgare L.) mutant, R5201, selected for resistance to 4? mM trans-4-hydroxyproline had a 3–6 fold increase in the soluble proline content of the leaf compared with the parent cultivar, Maris Mink. The mutant converted more [U-⁴C]glutamic acid to free proline in the leaves than Maris Mink but incorporation into protein proline was similar. Incorporation of radioactivity into proline was inhibited by exogenous proline more in Maris Mink than R5201, suggesting that feedback inhibition of proline biosynthesis is relaxed, but not absent in the mutant. When [1-¹⁴C]ornithine was the precursor, both R5201 and Maris Mink incorporated similar small amounts of label into soluble and protein proline. More protein proline was formed by both genotypes from labelled glutamic acid than from labelled ornithine. There may exist two routes of proline formation, where the glutamate pathway is synthetic and the ornithine pathway is catabolic.     

Changes in protein fractions and leucine-[14C] incorporation during Sorghum grain development

Incorporation of leucine and changes in different protein fractions have been studied during Sorghum grain development. Most of the label from the injected leucine-[¹⁴C] was found in glutelin and residue fraction towards later stages of maturity. The label in albumin, globulin and prolamin decreased with a concomitant increase in label in glutelin and residue proteins. The concentration of lysine, aspartic acid and glycine decreased while that of leucine, proline, alanine, tyrosine, phenylalanine, and cystine increased during grain development. Increase in serine, methionine, valine and isoleucine was only marginal. The proportion of glutamic acid was high at all stages of grain development. Glutelin fraction resolved into two peaks on gel chromatography, only one of which with higher MW was labelled, while in albumin both the peaks were found to be labelled. Tannin content also increased during grain development.     

Stoichiometry for guanine nucleotide binding to tubulin under polymerizing and nonpolymerizing conditions

The maximal stoichiometry for [³H]GTP binding to depolymerized tubulin with saturating amounts of added [³H]GTP is 0.4 mol/110,000 g protein. In contrast, 1 mol of radioactive nucleotide is incorporated into microtubules as a result of polymerization with [³H]GTP. The different stoichiometries result from a difference in the nucleotide binding properties of ring protein under polymerizing and nonpolymerizing conditions: ring protein at 0 °C is devoid of binding activity but binds added radioactive guanine nucleotide during microtubule assembly. The radioactive nucleotide which is incorporated into rings during microtubule assembly is not displaced by excess GDP, although it is at a site which is distinct from the N site.     

In vivo incorporation of [14C]tyrosine into the C-terminal position of the α subunit of tubulin

In vitro incorporation of [¹⁴C]tyrosine into the C-terminal position of the α subunit of tubulin was not affected by 4 mm cycloheximide. This inhibitor of protein synthesis was used for in vivo experiments. The in vivo incorporation of [¹⁴C]tyrosine into soluble brain protein of cycloheximide-treated rats was 10% of that of untreated rats. Treatment with vinblastine sulfate of the soluble brain protein showed that the incorporation of [¹⁴C]tyrosine into tubulin was higher in cycloheximide-treated than in untreated rats with respect to the incorporation into the total soluble protein. In the case of cycloheximide-treated rats, about 60% of the radioactivity incorporated into protein was released by the action of carboxypeptidase A, whereas 10% was liberated from the protein of untreated rats. The radioactive compound released by the action of carboxypeptidase A was identified as [¹⁴C]tyrosine. The α and β subunits of tubulin from animals that received [¹⁴C]tyrosine were separated by polyacrylamide gel electrophoresis. The radiosactivity ratio of $\text{α}\text{β}$ subunits of tubulin from cycloheximide-treated rats was threefold higher than that of untreated rats. When a mixture of [¹⁴C]amino acids was injected, the radioactivity ratio of $\text{α}\text{β}$ subunits of tubulin was similar for cycloheximide-treated and untreated rats. The results reported are consistent with the assumption that the α subunit of tubulin can be tyrosinated in vivo.     

AXOPLASMIC TRANSPORT IN THE OPTIC NERVE AND TRACT OF THE RABBIT

The axonal transport of labelled proteins was studied in the optic system of adult rabbits after an intraocular injection of [³H]Ieucine. It was demonstrated that the precursor was incorporated into protein, which was transported along the axons of the retinal ganglion cells. Intraocularly injected puromycin inhibited protein synthesis in the retina and markedly inhibited the appearance of labelled protein in the optic nerve and tract. It was further demonstrated by intracisternal injection of [³H]leucine that an intraocular injection of puromycin did not affect the local protein synthesis in the optic nerve and tract. Cell fractionation studies of the optic nerve and tract showed that the rapidly migrating component, previously described as moving at an average rate of 110-150 mm/day, was largely associated with the microsomal fraction. About 40 per cent of the total protein-bound radioactivity in this component was found in the microsomal fraction and about 15 per cent was recovered in the soluble protein fraction. Most of the labelled material moving at a rate of 1-5-2 mm/day was soluble protein. The specific radioactivity of this component was about ten times greater than that of the fast one. In the slow component about 50 per cent of the radioactivity was found in the soluble protein fraction and about 10 per cent of the radioactivity was recovered in the microsomal fraction. Radioautography demonstrated incorporated label in the neuropil structures in the lateral geniculate body as early as 4-8 hr after intraocular injection. The labelling of the neuropil increased markedly during the first week, and could be observed after 3 weeks.     

Further studies of the effect of L-canavanine on the tobacco hornworm, Manduca sexta.

Fifth instar Manduca sexta growth response to injected doses of canavanine was concentration-dependent over a range of 0·5 to 2·0 mg/g body weight. Twenty-four hr after injection of ¹⁴C-guanidinooxy-d,l-canavanine, M. sexta larvae incorporated approximately 3·6% of the labelled l-canavanine into protein of non-gut tissue. Adult M. sexta mortality was related to the level of injected canavanine over a range of 2 to 8 mg/g body weight. Injection of as little as 2 mg canavanine/g body weight caused hyperactivity in adult M. sexta. Arginine, able to negate the toxic effects of canavanine during larval growth, was only marginally capable of overcoming canavanine effects on larval-pupal ecdysis.     

Grazing of intertidal benthic foraminifera on bacteria: Assessment using pulse-chase radiotracing

Although foraminifera are a dominant component of many marine benthic communities, quantification of their predation on prokaryotes remains an experimental challenge. We have developed an approach that allows us to study grazing by adult specimens of the calcareous species Haynesina germanica and Ammonia beccarii, and the single-chambered agglutinated species Psammophaga sp., on bacteria (Halomonas sp.), pulse-chase-labelled with ³H- and ¹⁴C-Leucine. The bacterivorous ciliate Uronema sp. and flagellate Pteridomonas sp. were used as positive controls. The rate of release of ³H when protozoa were incubated with the labelled bacteria indicated the predator's grazing rate; the proportion of ¹⁴C found in the foraminiferal biomass and shell indicated the prey assimilation rate. All three foraminiferal species grazed bacteria at a rate of 3.2-5.7 ng C ind⁻¹ h⁻¹ depending on bacterial concentrations. About 23% of the biomass of the ¹⁴C-labelled prey was most likely assimilated into foraminiferal pseudopodia, 12% was expelled in dissolved waste material, about 62% was respired and only 0.1% was incorporated into the carbonate shell. Extracellular digestion associated with pseudopodia could explain the very low proportion of the labelled food assimilated in the cell body and the significant proportion located in pseudopodial networks. These experiments also suggest that very little of the carbon ingested by adult calcareous foraminifera is incorporated into the shell. However, we cannot conclude that diet has no influence on the stable isotope composition of the shell since none of our calcareous specimens grew new chambers during the experiments.     

Cuticular sclerotization in larval and adult locusts, Schistocerca gregaria

The sclerotization of both larval and adult cuticle from the desert locust, Schistocerca gregaria, has been studied by measuring the incorporation of radioactive dopamine and N-acetyldopamine into the cuticle. The results are compared with the degree of sclerotization of the cuticle and the amount of sclerotizing enzyme present. The various parts of the cuticle differ considerably with respect to the degree of sclerotization: in adult locusts the mandibles and the dorsal mesothoracic cuticle contain about twenty times as much cross-linking material per mg cuticle than is present in the abdominal tergites and sclerites.The degree of sclerotization in the various types of cuticle is apparently not determined by the amounts of sclerotizing enzyme present, and the rate at which radioactive dopamine or N-acetyldopamine is incorporated into the cuticle appears also to be unrelated to the amount of enzyme.The degree of sclerotization of the various parts of the cuticle from fifth instar larvae corresponds with the amounts of labelled dopamine which are incorporated during the first day after ecdysis, whereas there is no correlation between sclerotization and the amounts of labelled dopamine which are incorporated in older larvae. The degree of sclerotization of adult cuticle after 1 day corresponds to the incorporation of dopamine during the first day. When older animals are compared only little correlation is observed. The relative rates of sclerotization in the various parts of the cuticle must therefore change as the adult insect grows older.The changes in the incorporation pattern during the development of the locust are discussed in relation to the physiological control of the sclerotization process.     

In vitro synthesis of zein-like protein by maize polyribosomes.

Free and membrane-bound polyribosomes were isolated in an undegraded form from developing maize kernels. Translation of the membrane-bound polyribosomes $\text{in vitro}$ produced one main radioactive protein. This protein was soluble in 70% ethanol and had the same mobility in electrophoresis on sodium dodecyl sulfate-gels as a zein standard. The ratio of [¹⁴C] leucine to [¹⁴C] lysine incorporated into the 70% ethanol extractable protein was similar to the mole fraction ratio of these amino acids in zein. The zein-like protein may represent as much as 50% of the total protein synthesized by the membrane-bound polyribosomes.